This invention relates generally to the area of undifferentiated cells and methods of producing such cells. More specifically, the invention relates to pluripotent ungulate cells, particularly porcine cells, and to transgenic and chimeric ungulates produced from such cells.
Embryonic stem cells (ES cells) were first cultured from mouse embryos using a feeder layer of mouse fibroblasts or media conditioned with buffalo rat liver cells. The established ESC lines from mouse embryos have a characteristic phenotype consisting of a large nucleus, a prominent nucleolus, and relatively little cytoplasm. Such cells can be grown relatively indefinitely using the appropriate culture conditions. They can be induced to differentiate in vitro using retinoic acid or spontaneously by removal of the feeder layer or conditioned media. In addition, these cells can be injected into a mouse blastocyst to form a somatic and germ line chimera. This latter property has allowed mouse ESCs to be used for the production of transgenic mice with specific changes to the genome. See M. Evans et al., Nature 292, 154 (1981); G. Martin, Proc. Natl. Acad. Sci. USA 78, 7638 (1981); A. Smith et al., Developmental Biology 121, 1 (1987); T. Doetschman et al., Developmental Biology 127, 224 (1988)(; A. Handyside et al., Roux""s Arch Dev. Biol. 198, 48 (1989).
The active compound that allows the culture of murine embryonic stem cells has been identified as differentiation inhibiting activity (DIA), also known as leukemia inhibitory factor (LIF). See A. Smith, J. Tiss. cult. Meth. 13, 89 (1991); J. Nichols et al., Development 110, 1341 (1990). Recombinant forms of LIF can be used to obtain ESCs from mouse embryos. See S. Pease et al., Developmental Biology 141, 344 (1990). Also see U.S. Pat. No. 5,166,065 issued Nov. 24, 1992 to Williams, et al.
Subsequent to the work with mouse embryos, several groups have attempted to develop stem cell lines from sheep, pig and cattle. A few reports indicate that a cell line with a stem cell-like appearance has been cultured from porcine embryos using culture conditions similar to that used for the mouse. See M. Evans et al., PCT Application WO90/03432; E. Notarianni et al., J. Reprod. Fert., Suppl. 41, 51 (1990); J. Piedrahita et al., Theriogenology 34, 879 (1990); E. Notarianni et al., Proceedings of the 4th World Congress on Genetics Applied to Livestock Productions, 58 (Edinburgh, July 1990).
Attempts have been made regarding the culture of embryonic stem cells from avian embryos. It is difficult to establish a continuous line of chicken cells without viral or chemical transformation, and most primary chicken lines do not survive beyond 2-3 months. The culture of cells from the unincubated embryo is difficult, and under reported conditions such cells do not survive beyond two weeks. See E. Mitrani et al., Differentiation 21, 56-61 (1982); E. Sanders et al., Cell Tissue Res. 220, 539 (1981).
In U.S. Pat. No. 5,340,740 Petille et al. cultured chicken embryo cells on a mouse feeder layer in the presence of conditioned media and obtained the cultured stem cells.
Embryonic stem (ES) cells, the pluripotent outgrowths of blastocysts, can be cultured and manipulated in vitro and then returned to the embryonic environment to contribute normally to all tissues including the germline (for review see Robertson, E. G. (1986) Trends in Genetics 2:9-13). Not only can ES cells propagated in vitro contribute efficiently to the formation of chimeras, including germline chimeras, but in addition, these cells can be manipulated in vitro without losing their capacity to generate germ-line chimeras (Robertson, E. J., et al. (1986) Nature, 323:445-447).
ES cells thus provide a route for the generation of transgenic animals such as transgenic mice, a route which has a number of important advantages compared with more conventional techniques, such as zygote injection and viral infection (Wagner and Stewart (1986) in Experimental Approaches to Embryonic Development. J. Rossant and A. Pedersen eds. Cambridge; Cambridge University Press), for introducing new genetic material into such animals.
However, it is known that ES cells and certain EC (embryonal carcinoma) cell lines will only retain the stem cell phenotype in vitro when cultured on a feeder layer of fibroblasts (such as murine STO cells, e.g., Martin, G. R. and Evans, M. J. (1975) Proc. Natl. Acad. Sci. USA 72:1441-1445) or when cultured in medium conditioned by certain cells (e.g. Koopman, P. and Cotton, R. G. H. (1984) Exp. Cell Res. 154:233-242; Smith, A. G. and Hooper, M. L. (1987) Devel. Biol. 121:1-91). In the absence of feeder cells or conditioned medium, the ES cells spontaneously differentiate into a wide variety of cell types, resembling those found during embryogenesis and in the adult animal. The factors responsible for maintaining the pluripotency of ES cells have, however, remained poorly characterized.
The above methods involve the use of ES cells as starting materials. Very limited numbers of such cells are available. Any method which would allow for producing large numbers of ES cell would be very desirable.
A method of producing ungulate cells (porcine cells in particular) exhibiting an embryonic stem cell phenotype is disclosed as are the resulting pluripotent cells and chimeric ungulates (e.g., porcine) produced from the cells. Primordial germ cells are isolated from gonadal ridges of an ungulate embryo at a particular stage in development e.g., day-25 porcine embryos. The stage of development at which primordial germ cells are preferably extracted from an embryo of a particular species will vary with the species. For example, primordial germ cells are preferably extracted from a day 34-40 bovine embryo. Determination of the appropriate embryonic developmental stage for such extraction is readily performed using the guidance provided herein and ordinary skill in the art. The PG cells were cultured on inactivated STO cells under growth inducing conditions in long term cell culture (over 30 days). The resulting cells resembled ES cells in morphology including a large nucleus, prominent nucleoli and reduced cytoplasm as compared with differentiated adult cells. The cells can be passed several times in culture, be maintained for several months in culture, and survive cryopreservation in liquid nitrogen.
An object of the invention is to provide a method for producing ungulate cells (e.g., porcine cells) which exhibit an ES cell phenotype.
Another object is to provide pluripotent cells using germ cells as a starting material.
Another object is to provide chimeric ungulates (e.g., porcine, and bovine) using pluripotent cells of the invention.
Yet another object is to provide useful pharmaceutical products from the chimeric or transgenic ungulates produced with the cells of the invention.
An advantage of the invention is that large numbers of pluripotent cells can be quickly and efficiently produced from cells of an embryo thought to have developed too far to provide a source for pluripotent cells.
Another advantage is that the pluripotent cells of the invention can be used to produce a wide range of different chimeric ungulates (e.g., porcine) via homologous recombination methodology.
Yet another advantage of the invention is that thousands of pluripotent cells can be quickly and efficiently produced from germ cells extracted from a single ungulate embryo.
A feature of the invention is that the starting material is primordial germ cells isolated from gonadal ridges of ungulate embryo (e.g., day-25 porcine embryos or 34-40 day bovine embryos).
These and other objects, advantages, and features of the invention will become apparent to those persons skilled in the art upon reading the details of the ungulate cells expressing embryonic stem cell phenotype, method of making same, and chimeric and transgenic ungulates as more fully described below.
Before the present ungulate cells expressing embryonic stem cell phenotype, and methodology for making such ungulate cells are described, it is to be understood that this invention is not limited to particular cells, methods or chimeric ungulates described, as such cells, methods, and ungulates may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are now described. All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited.
The Publications discussed herein are cited for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the invention is not entitled to antidate such disclosure and the filing date by virtue of prior invention.
Definitions
The term xe2x80x9cungulatexe2x80x9d is used to mean any species or subspecies of porcine (pig), bovine (cattle), ovine (sheep) and caprine (goats). In general the term encompasses hooved farm animals.
The terms xe2x80x9cporcinexe2x80x9d and xe2x80x9cpigxe2x80x9d are used is interchangeably herein and refer to any porcine species and/or subspecies of porcine and the same meaning applies as to cows, sheep and goats.
The terms xe2x80x9cembryonic stem cell phenotypexe2x80x9d and xe2x80x9cembryonic stem-like cellxe2x80x9d are used interchangeably herein to describe cells which are undifferentiated and thus are pluripotent which cells are visually distinguished from other differentiated adult cells of the same animal e.g., by a noticeably larger nucleus, (25% or more larger), noticeably larger and prominent nucleolus and smaller (25% or smaller) cytoplasm as compared to differentiated adult cells of the same animal.
The term xe2x80x9cprimordial germ cellsxe2x80x9d is used to describe undifferentiated cells isolated from the gonadal ridges of an ungulate embryo (e.g. day-25 porcine embryo) or a 34-40 day bovine embryo.
The terms xe2x80x9cembryonic germ cellxe2x80x9d and xe2x80x9cgerm cell expressing ES cell phenotypexe2x80x9d are used to describe cells of the present invention which exhibit an embryonic stem cell phenotype.
The term xe2x80x9cembryonic stem cellxe2x80x9d is used to mean an undifferentiated cell isolated in its native form from the inner cell mass of a blastocyst-stage embryos, particularly blastocyst-stage porcine embryos at eight days or less after fertilization. Embryonic stem cells are pluripotent and have a noticeably larger nucleus, prominent nucleolus and smaller cytoplasm than adult cells of the same animal.
The term xe2x80x9cSTO cellxe2x80x9d refer to embryonic fibroblast mouse cells such are commercially available and include those deposited as ATCC CRL 1503, and ATCC 56-X.
The term xe2x80x9cchimericxe2x80x9d is used to describe an organism which includes genetic material from two different organisms. Specifically, a chimeric is produced by inserting cells of the invention which exhibit embryonic stem cell phenotype which cells were extracted from a first organism into early stage embryos (preimplantation embryos such as the blastocyst stage) of a second, different organism. The animal resulting from such methodology will include genetic material from the first and second organisms and thus be a xe2x80x9cchimericxe2x80x9d organism. Provided that the cell expressing embryonic stem cell phenotype is genetically manipulated to include exogenous material the resulting chimeric will include that exogenous material within some, but not all of its cells.
The term xe2x80x9ctransgenicxe2x80x9d is used to describe an animal which includes exogenous genetic material within its cells. Cells of the invention can have DNA added to them and these cells can then be used in a manner similar to that for making a chimeric animal. The resulting animal may be chimeric and transgenic. A xe2x80x9ctransgenicxe2x80x9d animal can be produced by intercrossing or backcrossing typically a chimeric male which include exogenous genetic material within cells used in reproduction to homozygosity. Twenty-five percent of the resulting offspring will be transgenic i.e., animals which include the exogenous genetic material within all of their cells in both alleles. 50% of the resulting animals will include the exogenous genetic material within one allele and 25% will include no exogenous genetic material.
Porcine Embryonic Stem-like Cells
Native primordial germ cells were isolated from gonadal ridges of day-25 porcine embryos. Cells may be isolated from anywhere on the dorsal mesentery provided the cells test positive for alkaline phosphate activity. The cell can be isolated at a time in the range of from about 23 to 27 days after fertilization. Cells outside of this range can be tested using the present invention to determine if desirable results can be obtained. In general, fewer cells are available for harvest earlier than 25 days and after 25 days the percentage of cells which exhibit pluripotency decreases. At the 25-day point about 10,000 cells exhibiting alkaline phosphatase activity (AP-positive) can be isolated from a given porcine embryo. The purity of the isolated cells will generally be in the range of about 70% to 90%.
Two to four thousand AP-positive cells per well were plated in 96-well tissue culture plates. The cells were cultured on inactivated STO cells at 39xc2x0 C. in an atmosphere of 5% CO2 in air.
All combinations of three different growth factors were used with the medium. The growth factors were leukemia inhibitory factor (LIF) at 1,000 units/ml stem cell factor at 60 ng/ml, and basic fibroblast growth factor at 20 ng/ml. Although the PG cells proliferated for 3 days and survived at least for 5 days in primary culture, none of the growth factors markedly induced proliferation of the PG cells during this limited period of time. However, when primordial germ cells extracted in the same manner were cultured over a much longer period of time (over 30 days) were assessed by both AP staining and morphology, the cells resembled ES cells in morphology and were AP positive. The use of LIF does not appear to be essential i.e., the growth medium can include only stem cell factor and basic fibroblast growth factor.
Undulate Embryonic Stem-like Cells
Other ungulates including cattle, sheep and goats can be manipulated in a manner similar to that described above with respect to porcine embryonic stem-like cells. First the particular ungulate is inseminated which insemination is preferably artificial for convenience. The embryo is extracted at a point in time wherein development approximately equals the 25-dayxc2x12 day porcine or 34-40 day bovine embryo. Specifically, the primordial germ cells must have accumulated at the beginnings of the formation of gonadal ridges but should not have been allowed to develop such that these cells become differentiated. Cells may be collected from the dorsal mesentery or gonadal ridge. For different animals the embryos can be extracted at different points in time and cells extracted from the gonad area of the embryo and tested using alkaline phosphatase activity (and morphology) as a positive test for cells which exhibit pluripotency. When AP-positive cells are isolated the cells from the same colony with the same morphology are cultured (e.g., on inactivated STO cells using appropriate conditions and culture medium). The cells must be cultured over long periods of time (over 30 days) in order to develop the desired cells expressing embryonic stem cell phenotype.
Chimeric Porcine
Cells produced by the methodology of the present invention are particularly useful in the preparation of chimeric animals which in turn can be bred to produce transgenic animals. Specifically, cells of the invention which exhibit embryonic stem cell phenotype can be genetically manipulated in a variety of different ways. For example, it is possible to use electroporation to insert a gene construct carrying a desired gene into these cells. After being genetically manipulated (to include exogenous DNA) the cells can be microinjected into a blastocyst of an ungulate of the same species e.g., porcine cell into a porcine blastocyst. That blastocyst is then placed into a pseudopregnant female porcine. The foster mother then carries the implanted blastocyst to term. Similar procedures with respect to mice are known. See M. Evans et al., Nature 292, 154 (1981); G. Martin, Proc. Natl. Acad. Sci. USA 78, 7638 (1981); A. Smith et al., Developmental Biology 121, 1 (1987); T. Doetschman et al., Developmental Biology 127, 224 (1988)(; A. Handyside et al., Roux""s Arch Dev. Biol. 198, 48 (1989). Also see U.S. Pat. No. 5,387,742, issued Feb. 7, 1995 to Cordell. These published procedures can be modified by those skilled in the art to apply to ungulates in general and specifically porcines, see published PCT Application WO94/26884 which is incorporated herein by reference in its entirety.
The chimeric porcine produced according to the above described process can be used for the production of any desired pharmaceutically active product. For example, the exogenous DNA could be a gene encoding human insulin which gene could be added to a cell of the present invention via electroporation. That cell containing the human insulin gene could then be included within a porcine blastocyst as described above to produce a chimeric porcine which would include cells which produce human insulin which can be extracted, purified and administered as a drug.
Chimeric Ungulate
Cells which exhibit embryonic stem cell morphology and are produced in accordance with the methodology of the present invention can be used to produce other chimeric ungulates including cows, sheep and goats. As indicated above, the cells exhibiting ES cell phenotype are genetically manipulated so that they incorporate exogenous genetic material. Typically the cells are subjected to electroporation to insert a vector carrying a desired gene. The genetically manipulated cells are then microinjected into the blastocyst of an ungulate of the same species as the germ cells were obtained. The blastocyst is inserted into a pseudopregnant ungulate female which is allowed to carry the blastocyst to term. Thus the invention includes inserting a manipulated ungulate cell which has embryonic stem cell phenotype characteristics and a first genetic complement into a host embryo (preferably at the blastocyst stage) of the same species from which the original germ cell was extracted. The host embryo has a second genetic complement which is generally different from the first genetic complement.
Utility
The methodology and cells of the present invention have a variety of different uses. In addition to being used to produce chimeric ungulates including porcine as described above the cells can be used to study embryological development. For example, the cells of the invention which exhibit embryonic stem cell phenotype can be genetically manipulated with labels or marker genes. The markers can then be inserted into blastocysts in order to observe distribution during the growth of the animal.
The embryonic ungulate stem cell may include an exogenous nucleotide segment which encodes any selectable marker. Examples of a suitable marker are hygromycin (Hph) (Yates et al., 1985), Neomycin (Neo) (Mansour et al., 1988, Nature 336:348-352) and puromycin (Pac) markers include ADA (adenosine deaminase) and dHFR (dihydrofolate reductase). A marker is useful to trace the cell lineage of linked transgenes of interest.
Some of the specific advantages of using the cells of the invention which exhibit embryonic stem cell phenotype are as follows. First, a gene of interest can be introduced and its integration and expression characterized in vitro. Secondly, the effect of an introduced exogenous gene on the ES cell growth can be studied in vitro. Thirdly, the characterized ES-like cells having a novel introduced exogenous gene can be efficiently introduced into embryos by blastocyst injection or embryo aggregation and the consequences of the introduced gene on the development of the resulting transgenic or chimeras monitored during pre- or post-natal life. Fourthly, the site in the ES cell genome at which the introduced gene integrates can be manipulated, leaving the way open for gene targeting and gene replacement (Thomas, K. R. and Capecci, M. R. (1987) Cell 51:503-512). See also U.S. Pat. No. 5,464,764 issued Nov. 7, 1995 to Capecci, et al. A gene can be ablated and the effect of such on the development of an ungulate studied over time.
Chimeric and transgenic animals are an alternative xe2x80x9cfactoryxe2x80x9d for making useful proteins by recombinant genetic techniques. Large animals such as pigs, cattle, sheep, and goats are potential factories for some products not obtainable from recombinant hosts such as microorganisms or small animals. Examples of such products are organs which are transplantable into humans.